Sphingosine, a Modulator of Human Translesion DNAPolymerase Activity*
Received for publication, March 31, 2014, and in revised form, June 12, 2014 Published, JBC Papers in Press, June 13, 2014, DOI 10.1074/jbc.M114.570242
Ashwini S. Kamath-Loeb‡1, Sharath Balakrishna‡1,2, Dale Whittington§, Jiang-Cheng Shen‡, Mary J. Emond¶,Takayoshi Okabe�, Chikahide Masutani**, Fumio Hanaoka‡‡, Susumu Nishimura§§, and Lawrence A. Loeb‡3
From the ‡Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University ofWashington, Seattle, Washington 98195, the §Department of Medicinal Chemistry, Mass Spectrometry Center, University ofWashington, Seattle, Washington 98195, ¶Biostatistics and Center for Biomedical Statistics, University of Washington, Seattle,Washington 98195, the �Open Innovation Center for Drug Discovery, University of Tokyo, Tokyo 113-0033, Japan, the**Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan,the ‡‡Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan, and the §§LaboratoryAnimal Resource Center, University of Tsukuba, Tsukuba 305-8575, Japan
Background: DNA polymerase � is a specialized, error-prone DNA polymerase capable of synthesis past bulky DNAadducts.Results: Sphingosine and sphinganine stimulate the activity of Pol �.Conclusion: Sphingosine modulates DNA lesion bypass in addition to controlling cell proliferation following DNA damage.Significance: There are no known stimulators of DNA polymerases. Stimulation by sphingosine represents a novel mode ofmodulating Pol � activity.
Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replica-tion-stalling DNA adducts. In so doing, they facilitate theprogression of DNA synthesis and promote cell proliferation.To potentiate the effect of cancer chemotherapeutic regi-mens, we sought to identify inhibitors of TLS DNA poly-merases. We screened five libraries of �3000 small mole-cules, including one comprising �600 nucleoside analogs, fortheir effect on primer extension activity of DNA polymerase� (Pol �). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol� by �100-fold at low micromolar concentrations but inhib-its it at higher concentrations. This effect is specific to theY-family DNA polymerases, Pols �, �, and �. The addition of asingle phosphate group on sphingosine completely abrogatesthis effect. Likewise, the inclusion of other sphingolipids,including ceramide and sphingomyelin to extension reac-tions does not elicit this response. Sphingosine increases therate of correct and incorrect nucleotide incorporation whilehaving no effect on polymerase processivity. Endogenous Pol� activity is modulated similarly as the recombinant enzyme.Importantly, sphingosine-treated cells exhibit increasedlesion bypass activity, and sphingosine tethered to membranelipids mimics the effects of free sphingosine. Our studies haveuncovered sphingosine as a modulator of TLS DNA polymer-ase activity; this property of sphingosine may be associated
with its known role as a signaling molecule in regulating cellproliferation in response to cellular stress.
TLS4 DNA polymerases are an evolutionarily conserved fam-ily of specialized, error-prone DNA polymerases. They are dis-tinguished from other DNA polymerases by the presence ofcapacious active site binding pockets that can accommodateand enable DNA synthesis past bulky DNA adducts (1–3). In sodoing, TLS DNA polymerases help prevent the stalling and col-lapse of replication forks and ensuing DNA breaks/rearrange-ments. The largest class of such DNA polymerases is the Y-fam-ily, which includes, in human cells, Pols �, �, and �, and Rev1.DNA Pol � has garnered the most attention as mutations inPOLH are causally linked to the variant form of Xerodermapigmentosum (4), an inherited disease associated with sunlightsensitivity and increased incidence of skin cancers (5). Pol � hasbeen shown in vitro to efficiently and accurately bypass theUV-induced lesion, cyclobutane pyrimidine dimer (CPD) (6). Itis believed that in the absence of Pol �, error-prone translesionsynthesis across CPD lesions by other specialized DNA poly-merases, including Pol �, results in increased mutagenesis andcarcinogenesis. Pol � has also been shown to bypass cisplatin-induced cross-linked adducts (7) and oxidative lesions (8). Inaddition, by virtue of its error-prone nature, Pol � has beenimplicated in immunoglobulin gene somatic hypermutation(9).
Although no specific disorders are associated with the otherY-family TLS DNA polymerases, each of them has unique attri-butes. Pol �, an ortholog of Pol �, is the only polymerase that is
* This work was supported, in whole or in part, by National Institutes of HealthGrants P01-CA 077852 and P01-AG 001751 (to L. A. L.).
1 Both authors contributed equally to this work.2 Present address: Div. of Genomics, Dept. of Cell Biology and Molecular
Genetics, Sri Devaraj Urs Academy of Higher Education and Research, Kolar563101, Karnataka, India.
3 To whom correspondence should be addressed. Tel.: 206-543-6015; Fax:206-543-3967; E-mail: [email protected].
4 The abbreviations used are: TLS, translesion; Pol �, DNA polymerase �; SA,sphinganine; SO, sphingosine; S1P, sphingosine-1-phosphate; SM, sphin-gomyelin; CPD, cyclobutane pyrimidine dimer; P, primer; T, template;qPCR, quantitative PCR; UPLC, ultra high-pressure liquid chromatography.
reported to use Hoogsteen base pairing to efficiently mispairdG residues across template dT (10). Pol � specializes in thebypass of benzo[a]pyrene diol-epoxide-DNA adducts; it copiespast these lesions in vitro, and cells depleted for Pol � are sen-sitive to benzo[a]pyrene diol-epoxide (11, 12). Additionally, ithas been reported that mutagenic bypass activity by Pol �homologs (DinB) consists predominantly of �1 frameshifts,which arise from the extra-helical displacement of DNA lesions(13). REV1, or deoxcytidyl transferase, is restricted to insertingdC residues opposite template lesions, including abasic sitesand N2-dG adducts (14). An additional non-catalytic, scaffold-ing role for REV1 that facilitates polymerase switch during TLShas also been proposed (15).
Due to the mutagenic nature of DNA synthesis by TLS DNApolymerases, cells employ an array of regulatory mechanisms tolimit TLS activity, including post-translational modifications,relocalization upon DNA damage, and binding to accessoryproteins (1, 13). Some cancer cells take advantage of error-prone TLS to bypass DNA adducts generated by chemothera-peutic drugs (16). One manner in which they do so is by up-reg-ulating the level of TLS DNA polymerases. For example, Pol �levels are reported to be elevated in lung cancer (17), whereasPol � levels are increased in breast cancer (18). In addition, cis-platin or oxaliplatin treatment has been shown to induce Pol �in non-small-cell lung cancer and gastric adenocarcinoma (19,20). Overexpression of translesion DNA polymerases mayreduce the effectiveness of chemotherapy regimens and rendercancer cells resistant to chemotherapy (21). Consistent withthis, Doles et al. (22) and Xie et al. (23) demonstrated that sup-pression of REV3, the catalytic subunit of the B-family TLSpolymerase Pol �, sensitizes drug-resistant lung tumors tochemotherapy.
We screened several collections of small molecule com-pounds to identify inhibitors of TLS DNA polymerases thatcould potentiate the effect of cancer chemotherapeutic agents.We identified and report here on the surprising discovery of amodulator, which both stimulates and inhibits TLS DNA poly-merases. We identified the compound as the biologically activesphingolipids, sphingosine, and dihydrosphingosine (sphinga-nine). We show that of all sphingolipids evaluated, sphingosineand sphinganine alone manifest this effect. Although low con-centrations markedly stimulate the rate of DNA primer exten-sion as well as the extent of nucleotide misincorporation andmisextension by Pol �, high concentrations inhibit polymeraseactivity. These effects of sphingosine are specific to the Y-familyTLS polymerases, Pols �, �, and �. Notably, sphingosineincreases lesion bypass activity in cells, and sphingosine boundto model liposomes manifests similar effects as the free com-pound. Our studies have uncovered a novel mode of modula-tion of Pol � and have linked two responses of cells to stress,namely DNA lesion bypass by TLS DNA polymerases andsphingolipid-mediated signaling to control cell proliferation.
EXPERIMENTAL PROCEDURES
Reagents and Enzymes—Five small molecule libraries wereanalyzed; four were obtained from the National Institutes ofHealth (NCI-approved Oncology Drug Set, Mechanistic Set,Diversity Set II, and Natural Product Set II), and one consisting
of 622 compounds enriched for nucleoside analogs, was fromthe Open Innovation Center for Drug Discovery, The Univer-sity of Tokyo, Japan. The compounds were stored as 10 mM
stock solutions in dimethyl sulfoxide at �80 °C. D-erythro-Sphinganine, D-erythro-sphingosine, D-erythro-sphingosine-1-phosphate, and natural brain ceramide and sphingomyelinwere purchased from Avanti Polar Lipids Inc (Alabaster, AL).L-erythro-Sphingosine was obtained from Sigma-Aldrich. Gel-purified synthetic oligonucleotides were obtained from Inte-grated DNA Technologies (Coralville, IA). The sequence of theprimer-template (P/T1) used for measuring the effect of smallmolecules on DNA polymerase activity was 5�-CGCGC-CGAATTCCCGCTAGCAAT-3� and 5�-GCGCGGAAGCT-TGGCTGCAGAAGATTGCAGCGGGAATTCGGCGCG-3�.The sequence of the primer-template (P/T2) used for evalu-ating CPD bypass activity in vitro was 5�-CACTGACTG-TATGA-3� and 5�-CTCGTCAGCATCT-TCATCATACAG-TCAGTG-3�, whereas that (P/T3) used for measurements ofVmax and Km values was 5�-CACTGACTGTATGATG-3� and5�-CTCGTCAGCATCT-TCATCATACAGTCAGTG-3�,where T-T indicates the cyclobutane pyrimidine dimer. TheT-T DNA template was generously provided by S. Iwai (OsakaUniversity, Osaka, Japan). A template containing two consecu-tive T residues in place of the CPD lesion was used in controlreactions.
Human DNA polymerase � (1–511 amino acids) was purifiedfrom insect cells as described (6). Full-length Pols �, �, and �were purchased from Enzymax (Lexington, KY). Human Pols �and � were expressed and purified as described (24),5 whereashuman Pol � was purified from baculovirus-infected insectcells.6 Calf thymus Pol -primase complex and human Pol were generous gifts from F. Perrino (Wake Forest Park, NC)and S. Linn (Berkeley, CA), respectively.
Sample Fractionation and Mass Spectrometry—Samplescontaining the modulator were separated on an Agilent 1290Infinity System. A Thermo Hypersil Gold UPLC column cou-pled to a C18 guard cartridge was used for fractionationemploying a gradient of water and methanol as the mobilephase. Fractions were collected at 1-min intervals for a totalduration of 20 min, dried under N2, resuspended in dimethylsulfoxide, and evaluated in polymerase activity assays. Peakfractions were subject to a second round of ultra high-pressureliquid chromatography (UPLC) fractionation; the identity ofthe compound in the single fraction that tested positive forstimulatory activity was determined by mass spectrometryusing a Waters Synapt G1 QTOF instrument.
Primer Extension Assays—5�-32P-labeled DNA primers (P)were hybridized to a 2-fold molar excess of complementarytemplate (T) DNA. 10 nM of the P/T DNA substrate wasextended by limiting amounts (�0.2 nM) of DNA polymerase �in the absence or presence of the indicated sphingolipid at 37 °Cfor 10 min, unless stated otherwise. Reactions were carried outin buffer containing 40 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 10mM DTT, 60 mM KCl, and 2.5% glycerol. Control reactions forsphinganine or sphingosine (SO) contained 10% dimethyl sulf-
5 J.-C. Shen, unpublished data.6 M. W. Schmitt, unpublished data.
Sphingosine Modulates Human TLS DNA Polymerase Activity
21664 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014
oxide, whereas those for ceramide, sphingomyelin, or sphingo-sine-1-phosphate (S1P) included 10% methanol. Aliquots of theterminated reactions were electrophoresed through 14% poly-acrylamide-urea gels; extension products were visualized byPhosphorImager analyses (GE Healthcare) and quantifiedusing ImageJ software (NIH).
Measurements of the kinetic constants for single nucleotideincorporation by Pol � were carried out as described (25).Briefly, we first systematically varied the enzyme concentrationand reaction time using saturating concentrations of dNTPs.These experiments established the requisite conditions forsubsequent kinetic analysis, i.e. linearity of incorporationwith time during a fixed incubation period. Extension wasthen monitored as a function of dNTP and sphingosineconcentrations.
Immunoprecipitation—� 2 � 107 human embryonic kidneyepithelial cells (293T) were lysed by incubation in 500 �l of lysisbuffer (200 mM Hepes-KOH, pH 7.4, 155 mM KCl, 1.5 mM
MgCl2, 2 mM DTT, 0.5% Triton X-100, 0.2 mM PMSF, and 10�g/ml of aprotinin, pepstatin, and leupeptin) on ice for 10 min.The suspension was centrifuged at 2000 � g for 10 min, and theclarified supernatant was used for immunoprecipitating Pol �;�500 �g of total protein were incubated with the Pol �-specificmonoclonal antibody, 5C6 for 1 h at 4 °C (26). A 25% suspen-sion of Protein A/G agarose beads was added to precipitate thecomplex, and incubation was carried out as described above.The enzyme-antibody complex coupled to beads was washedextensively with buffer containing 20 mM Tris-HCl buffer, pH7.5, 150 mM NaCl, 1% Nonidet P-40, and 10% glycerol, andresuspended in 15 �l of 25 mM Tris-HCl buffer, pH 8.0, 0.5 mM
EDTA, 1 mM DTT, 0.05% Nonidet P-40, and 25% glycerol. Ali-quots of the resuspended immune complex were diluted andassayed for CPD bypass activity in the absence or presence of 8�M sphingosine.
CPD Bypass Activity in Cells—CPD bypass activity in cellswas measured using a modification of our recently establishedoligonucleotide retrieval assay (27, 28). A synthetic oligonu-cleotide containing a site-specific CPD lesion was extendedby ligating oligonucleotides, 5�-AmMC6ACGGAGGGAATC-GGAGGTCGC-3� and 5�-CCTTCCACCTCCCATTCCTGA-TTCAGTCACTG-ddC-3�, at the 5� and 3� termini, respec-tively. Complementary oligomers to form double-strandedligation sites at the 5� and 3� termini were 5�-TGCTGACGAG-GCGACCTCCG-3� and 5�-AGGTGGAAGGCACTGACTGT-3�, respectively. After ligation, excess free oligomers weredegraded by � exonuclease. The ligation efficiency (20%) wascalculated from the ratio of amplification of the ligated versusunligated template and used to normalize substrate concentra-tions. The template was then hybridized to a biotinylatedprimer, 5�-biotin-GCACGTCAGGCACGGCGTCCAGTGA-CTGAATCAGGAATGGGAGGTGGAAGGCACTGACTG-TATGATG-3�, to form the partial duplex DNA substrate.
This construct was transfected into SV40-immortalizedfibroblast cells (GM0639) using calcium phosphate; the finalconcentration was 0.2 nM. After incubation for 2 h, oligonucleo-tides were retrieved via magnetic streptavidin bead capture byvirtue of the 5�-biotin tag on the primer strand. Bypass effi-ciency was quantified by qPCR using the Brilliant III Ultra-Fast
SYBR Green qPCR Master Mix (Agilent) in a DNA EngineOpticon 2 machine (MJ Research). The PCR primer pairs formeasuring unextended and extended/bypassed productswere a/b (5�-GCACGTCAGGCACGGCGTC-3� and 5�-CATCATACAGTCAGTGCCTTCCACCTCC-3�) and a/c(5�-GCACGTCAGGCACGGCGTC-3� and 5�-GGAGGTC-GCCTCGTCAGCATC-3�), respectively. % Bypass was cal-culated as the ratio of (amplification by primers a/c overprimers a/b) � 100.
To test for a difference in CPD bypass activity in SO-treatedversus control cells, the experiment effect (shift in overallmeans on different experimental days seen in Fig. 5) andunequal variance among the different experiments were takeninto account using a log transformation of the outcome withweighted least squares regression (29), including a variable forexperiment in the model. Specifically, % bypass activity valueswere log-transformed, the variance of the transformed obser-vations was calculated within each experiment, and this vari-ance was used to inversely weight the observations in the leastsquare regression. Variables for both treatment (variable ofinterest) and experiment (confounder) were included in theweighted least squares regression model. This resulted in nor-mally distributed residuals as required for an unbiased test sta-tistic with this small sample size.
Liposome Preparation—Liposomes were generated by using2.5 mg of the synthetic phospholipid, 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (99% pure; Avanti Polar Lipids)either by itself or mixed with sphingosine or sphingomyelin at20 mol % with respect to 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine. Lipids, dissolved in chloroform or methanol,were dispensed in glass tubes, and the organic solvents werefirst evaporated under a stream of argon and further driedunder vacuum. Lipid films were hydrated by the addition of0.25 ml of 1� Pol � reaction buffer. The lipid suspension wasextruded through a 100-nm polycarbonate filter at roomtemperature (using the lipid extruder from Avanti Polar Lip-ids) to yield homogenous unilamellar vesicles. The vesicleswere stored at 4 °C and used within 3 days of preparation.Fraction SO and SM bound to liposomes (f) was calculatedusing the formula, f � K � Cl/1 � K � Cl, where K � 1/CMC(critical micelle concentration), and Cl is the lipid concen-tration; we estimate that � 96% of SO and SM were bound toliposomes.
RESULTS
Identification of the Modulator—We screened multiplelibraries of small molecules for inhibitors of DNA polymerasesas potential anti-tumor agents. To facilitate screening, wepooled the compounds into groups of four and evaluated theireffects on the polymerase activity of DNA Pol � by monitoringthe extension of an end-labeled primer. We identified one poolfrom the library of nucleoside analogs that markedly stimulatedthe polymerase activity of Pol �. Upon deconvolution, the stim-ulatory compound was identified as 6-chloropurine-tetrahy-dropyran (2-(6-aminopurin-9-yl)-6-(hydroxymethyl)-2H-3,4,5,6-tetrahydro-pyran-3,4,5-triol). This compound wasobtained from two independent commercial sources. Massspectrometric and NMR analyses revealed that one of the two
Sphingosine Modulates Human TLS DNA Polymerase Activity
AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21665
preparations was homogenous but lacked stimulatory activity.However, the second preparation that stimulated Pol � activityhad multiple components (Fig. 1A). To separate the stimulatory
factor from other components, we fractionated the secondpreparation by chromatography through two sequentialreverse-phase UPLC columns and assayed individual fractions
FIGURE 1. Mass spectrometric analyses of the stimulator. The compound (from the library of nucleoside analogs) containing stimulatory activity wasenriched by fractionation through two reverse phase ultra-pressure columns. Individual fractions were assayed for their effect on the polymerase activity ofDNA Pol �. Shown are the mass spectrometric profiles of the starting compound (A), the single fraction obtained after two rounds of UPLC purification thatstimulated Pol � activity (B), and the C17-sphinganine standard (C). Note the presence of the peak (arrow) with mass 288.2902, corresponding to that ofC17-sphinganine, which increased in abundance following UPLC (B). The scales used for the mass spectrometric profiles in A and B are 1.48 � 104 and 2.61 �105, respectively. Peaks other than that corresponding to mass 288.2902 in B were also present in the mobile phase.
Sphingosine Modulates Human TLS DNA Polymerase Activity
21666 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014
for their effect on Pol � activity. A single fraction containing acompound with an m/z of 288.2902 (Fig. 1B) stimulated primerextension by Pol � (data not shown). This mass did not corre-spond to that of the starting compound (expected mass, 238.7).A search of the Scripps-Metlin database for molecules with amass of 288.2902 5 ppm offered three possibilities, two ofwhich were deuterium compounds; the third was the sphingo-lipid, C-17 sphinganine (SA; Fig. 1C).
Sphinganine Uniquely Stimulates the Primer Extension Activ-ities of DNA Pols �, �, and �—To evaluate the effect of the identifiedsphingolipid, homogenous preparations of C-17 sphinganine wereadded to primer extension reactions. Surprisingly, we observedthat it stimulated Pol � activity; the increase in nucleotide incor-poration was linear with respect to SA concentrations from 1 to 10�M; primer extension was enhanced by �100-fold (Fig. 2A). Athigher concentrations, however, the activity declined precipi-tously. The decrease in activity could be related to the criticalmicelle concentration, reported to be in the low �M range (30),
which could block the accessibility of the enzyme active site todeoxynucleotide substrates and/or the DNA primer-template.Stimulation was indifferent to the order of addition of Pol � and SAand was comparable with both truncated and full-length Pol �,indicating that the C-terminal 202 amino acids, which include theproliferating cell nuclear antigen-binding motifs, are dispensablefor stimulation by SA (data not shown). Furthermore, stimulationwas independent of DNA sequence context, occurring with a mul-titude of primer-template DNA substrates, including templatescontaining cyclobutane pyrimidine dimers (data not shown andsee Fig. 5A). Notably, the stimulatory effect was specific to Pol �and to Pols � and �, members of the Y-family DNA polymerases.Although the extent of stimulation of Pol � paralleled that of Pol �,it was approximately an order of magnitude lower with Pol �. Incontrast, SA did not stimulate the primer extension activities of anarray of other mammalian DNA polymerases, including nuclearand mitochondrial replicative DNA polymerases, Pols , �, �, �,and (Fig. 2B).
FIGURE 2. Sphinganine and sphingosine uniquely stimulate the extension activity of Y-family TLS DNA polymerases, Pols �, �, and �. Homogenouspreparations of sphinganine were used at the indicated concentrations in primer extension reactions with Pol � (A) or at 10 �M concentrations with Pols �, �,�, , �, �, �, and (B). SA (10 �M), SO (0.5, 2, 10, and 30 �M); SM (0.5, 2, 10, 50, and 100 �M); ceramide (0.5, 2, 10, 50, and 100 �M); and S1P (0.7, 2.8, 14, 70, and 140�M) were evaluated for specificity in primer extension reactions with Pol �, (C). Control reactions for SO and SA contained 10% dimethyl sulfoxide, whereasthose for SM, ceramide (Cer), and S1P included 10% methanol. Extension reactions and gel electrophoresis were carried out as described under “ExperimentalProcedures.” S, substrate (�) enzyme.
Sphingosine Modulates Human TLS DNA Polymerase Activity
AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21667
Pol � Activity Is Stimulated by Sphingosine, but Not by OtherSphingolipids—Sphinganine (dihydrosphingosine) is identicalto SO except for a single saturated bond. Because the commonform of SO has an 18-carbon chain length, we examined theeffect of C-18 SO on Pol � activity in this and all subsequentexperiments carried out with SO. Similarly to C-17 SA, C-18 SOalso elicited the dual response by Pol � (Fig. 2C and data notshown); fold increase and peak stimulatory concentration ofSO, both D- and L-stereoisomers, were equivalent to that of SA.In addition, similarly to SA, it inhibited polymerase activity atconcentrations exceeding 10 �M. SA and SO are involved in thede novo synthesis of ceramide and production of S1P, respec-tively (31). In contrast to SO, neither ceramide nor S1P, even at10 –50-fold higher concentrations than SA and SO, exhibitedstimulatory or inhibitory effects on DNA synthesis by Pol �(Fig. 2C). Furthermore, the robust stimulation observed withSO was unaffected when it was present together with S1P at a1:1 ratio, suggesting that S1P likely does not interact function-ally or interfere with interactions between SO and Pol � (datanot shown). Free SO in the cell is generated from the hydrolysisof sphingomyelin (32). Consistent with the stimulatory effect ofSO, we observed subtle yet reproducible stimulation of Pol �activity by sphingomyelin (Fig. 2C). A short chain (C4) deriva-tive of SO, threoninol, had no effect on the activity of Pol � (datanot shown), implicating the importance of both the carbonchain length as well as the functional head group of SO in elic-iting the response by Pol �. As a control, we also included thenon-ionic detergent, Triton X-100 in extension reactions.Again, we observed no stimulation or inhibition of Pol � activity(data not shown), further corroborating the specificity of SAand SO.
Sphingosine Increases the Rate of Nucleotide Incorporation byPol � and Is Non-competitive with dNTP Binding—Kineticstudies were carried out to interpret the basis of stimulation.Processivity experiments, which evaluate the number of nucle-otides incorporated before the polymerase disengages from theDNA, revealed that Pol � is distributive. It incorporatesbetween 4 –5 nt at each nucleotide addition step; this remained
unchanged upon the addition of sphingosine, suggesting thatstimulation by SO is not the result of increased processivity byPol � (data not shown). We also measured the rate of incorpo-ration of the initiating dNTP as a function of dNTP and sphin-gosine concentration. Incorporation kinetics were measured inthe presence of increasing concentrations of dCTP (0.5–10 �M)without or with, 2, 4, or 8 �M SO. As presented in Fig. 3 andTable 1, there was a concentration-dependent increase in therate of incorporation of the initiating nucleotide with a 3-foldincrease in the kcat value with 8 �M SO. A similar analysis, whichexamined the rate of incorporation of dATP across a CPDlesion, also showed an elevated (5-fold) kcat upon the addition ofSO (data not shown). However, we did not observe changes inthe Km for dCTP in the presence of SO, consistent with SOinteractions being non-competitive with respect to dNTP bind-ing. SO could bind within a hydrophobic pocket of Pol � toinduce conformational changes that render the enzyme moreproficient. Attempts to visualize direct SO-Pol � interactionsby x-ray crystallography, however, have been unsuccessful thusfar.7
Sphingosine Increases Misincorporation of dNTPs—To exam-ine whether SO affects primer extension when one or moredNTPs is absent, reactions were carried out either in the pres-ence of single dNTPs, or with three of the four dNTPs. In single
7 W. Yang, personal communication.
FIGURE 3. Kinetic analysis of single nucleotide incorporation by Pol �. Steady state reactions to measure kinetic constants were carried out as describedunder “Experimental Procedures.” A representative gel (A) and the corresponding Michealis-Menten graphs (B) show the incorporation of the correct nucle-otide, dCTP (concentrations ranging from 0.5–10 �M) by Pol � as a function of the indicated concentrations of SO. 9, 6, and 4.5 fmol of Pol � were used inreactions with 0, 2, and 4/8 �M SO, respectively.
TABLE 1Steady-state kinetic parameters for nucleotide incorporation in theabsence or presence of sphingosineKm and kcat values for the incorporation of dCTP across from template dG by Pol �were measured under steady state conditions as described under “ExperimentalProcedures.” Reactions were carried out at 37 °C for 5 min in the absence or pres-ence of indicated concentrations of sphingosine. Data were fit by nonlinear regres-sion using GraphPad Prism software (version 5). Data are the average S.D. of twoto three independent measurements.
nucleotide addition experiments, SO stimulated the incorpora-tion of both the correct nucleotide, dCTP, and the incorrectnucleotides to comparable extents (Fig. 4A; 4- to 7-foldincrease). Steady-state reactions examining nucleotide misin-corporation kinetics also revealed an increase (2-fold) in themedian kcat value in the presence of 10 �M SO, with no changein the median Km value (data not shown). Similarly, SO stimu-lated extension reactions in the presence of three dNTPs.Strong pause sites were observed immediately preceding or atthe position corresponding to the missing nucleotide; however,in every case, read-through products were readily discernible inthe presence of SO, accounting for an �90% increase comparedwith reactions lacking SO (Fig. 4B). In preliminary experiments,we again observed a 2-fold increase in the rate of extension of a3�-terminal mismatched nucleotide (data not shown). Byenhancing the rate of nucleotide misincorporation and misex-tension, SO can likely increase the rate of mutagenesis by Pol �.
Sphingosine Stimulates Primer Extension Activity of Endoge-nous Pol �—To ascertain that native Pol � responds to SO sim-ilarly as the recombinant protein, we isolated Pol � from humancells by using a Pol �-specific monoclonal antibody and assayedprimer extension activity with the immunoprecipitatedenzyme. To ensure that the observed activity is due to Pol �, weassayed TLS across a CPD lesion in the absence or presence ofSO. We observed efficient thymine dimer bypass activity in theimmunoprecipitated sample, which was concentration-depen-dent (data not shown); this activity of endogenous Pol �increased upon the addition of SO (Fig. 5B), suggesting that Pol� can be regulated by SO in vivo. The magnitude of stimulationof native Pol � was not as high as that observed with the recom-binant protein (Fig. 5A); this could be due to the presence ofendogenous SO in the immunoprecipitate (data not shown).
Sphingosine Increases Lesion Bypass Activity in Cells—Toexamine whether SO increases translesion activity in cells, we
carried out initial experiments with a partial duplex DNAoligonucleotide construct containing a site-specific CPD lesion.We transfected this substrate into SV40-immortalized fibro-blasts (27, 28) and monitored bypass activity in the absence orpresence of SO treatment by qPCR, as described under “Exper-imental Procedures.” The apoptotic response triggered by SOprecluded the use of high concentrations. Nonetheless, cellstreated with low concentrations (2–5 �M) of SO exhibited 28%higher bypass activity compared with untreated cells; these dif-ferences in SO-treated samples were consistent across three
FIGURE 4. SO increases nucleotide misinsertion and misextension by Pol �. Pol � (0.6 nM) was incubated with DNA P/T1 (10 nM) in the presence of singledNTPs (either correct (dCTP) or incorrect (dATP, dGTP, dTTP) (A)) or three of four dNTPs (B), each at 100 �M concentrations. Extension reactions were carried outat 37 °C for 10 min in the absence (�) or presence (�) of 8 �M sphingosine. Note the strong pause sites at or immediately preceding the missing dNTP. S,substrate (�) enzyme; N, control with all four dNTPs. The sequence of the DNA template is shown on the left.
A
SO - - + - +CPD - - - + +
B
SO - - +S IP
T-T T-T
FIGURE 5. SO stimulates bypass activity of Pol �. CPD bypass activity ofrecombinant Pol � (A) or endogenous native enzyme immunoprecipitatedfrom human cells in culture (B) was monitored using P/T2 DNA substrate inthe absence or presence of 8 �M sphingosine as described under “Experimen-tal Procedures.” The control template contained two tandem dT residuesinstead of the lesion; the sequence was identical otherwise. S, substrate (�)enzyme.
Sphingosine Modulates Human TLS DNA Polymerase Activity
AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21669
independent experiments (p � 0.046 (95% confidence interval,2– 62% higher) (Fig. 6). While low, the fold increase observed inour experiments is as follows: a) statistically significant, b) con-sistent across three independent experiments, and c) similar inmagnitude to the increase in overall DNA synthesis observed byZhang et al. (33) in SO-treated cells.
Membrane-bound Sphingosine Mimics the Effects of FreeSphingosine—SO is a natural constituent of cells. It could betethered to membrane lipids in vivo via its hydrophobic alkylchain. To determine whether membrane-bound SO modulatesPol � activity similarly as unbound SO, we prepared modelunilamellar lipid vesicles using the synthetic phospholipid,phosphatidylcholine. Independent preparations containedeither SO or SM. The liposome preparations were diluted suchthat the concentrations of membrane-bound SO and SM werecomparable with those of free SO and SM used in experimentsthus far, and their effects were evaluated in primer extensionreactions containing Pol �. Membrane-bound SO, but not PCvesicles or membrane-bound SM, specifically modulated Pol �activity (Fig. 7). Membrane-bound SO both stimulated (Fig. 7A)and inhibited (Fig. 7B) DNA extension activity, albeit to a lowerextent than observed with free SO. Furthermore, the concen-trations that elicited this response were equivalent to those offree SO. For example, whereas 10 �M bound-SO stimulatedextension activity, 30 �M and higher concentrations elicitedstrong inhibition. We estimate that �96% of SO is membrane-bound under our conditions, and we have shown that concen-trations corresponding to 4% free SO have no effect on TLSpolymerase activity. Thus, both the specificity and responseelicited by SO in solution are recapitulated with membrane-bound SO.
DISCUSSION
We set out to identify small molecule inhibitors of TLS DNApolymerases. Instead, we serendipitously uncovered two
FIGURE 6. Sphingosine increases CPD lesion bypass activity in cells.GM0639 cells were transfected with a partial duplex DNA oligonucleotideconstruct containing a site-specific cyclobutane pyrimidine dimer. Followinga 2-h incubation period, the oligonucleotide was retrieved, and the extent oflesion bypass was quantified by qPCR using the primer pairs, a/b and a/c, asshown on the schematic. % Bypass observed in untreated (�) or SO-treated((�); 2 �M (experiment 1) or 5 �M (experiments 2 and 3)) cells in three inde-pendent trials is presented. ddC, dideoxycytidine; AmMC, 5�-amino modifierC6.
FIGURE 7. Membrane-bound SO mimics the effect of free SO. Liposomes prepared with 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine, either alone ormixed with SO or SM, were added at increasing concentrations to primer extension reactions to evaluate stimulatory (A) or inhibitory (B) effects. Visualizationof maximal stimulation and inhibition by liposomes with SO necessitated the use of different Pol � amounts in the extension reactions presented in A and B. Pol� (2 fmol) was incubated with 3, 10, or 30 �M liposomes (Lipo) or liposomes with SO (Lipo-SO) or with 10 or 100 �M liposomes with sphingomyelin (Lipo-SM) inA, or 9 fmol of Pol � were incubated with 0.01, 0.1, 1, 3, 10, 30, or 100 �M of each liposome preparation in B. Primer extension reactions with P/T1 were carriedout as described, and products were visualized by PhosphorImager analysis of denaturing gels. S, substrate (�) enzyme.
Sphingosine Modulates Human TLS DNA Polymerase Activity
21670 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014
related stimulators, the lipid-signaling molecules, sphingosineand dihydrosphingosine. Only about a dozen mechanisticallywell-characterized enzyme activators have been identified thusfar, but none of them target DNA metabolic enzymes, let aloneDNA polymerases (34). To our knowledge, sphingolipids repre-sent the first example of naturally occurring lipid compounds thatmarkedly stimulate DNA polymerase activity and, uniquely, thatof the Y-family TLS DNA polymerases, Pols �, �, and �.
Sphingosine, the simplest of all sphingolipids, is recognizedfor its role in cell signaling to control cell proliferation (31,35–37). Our studies, however, have uncovered a hithertounknown and unreported function of sphingosine, namely, itsability to stimulate the DNA polymerase activity of Y-familyTLS DNA polymerases. We show that SA and SO, but not thephosphorylated form S1P or other sphingolipids, stimulate invitro the DNA primer extension activity of TLS DNA poly-merases in a sequence-independent manner (Figs. 2 and 5A).Stimulation is specific to Pols �, �, and �; the polymerase activ-ities of family A, B, and X DNA polymerases are unaffected (Fig.2, B and C). The effect of SO is non-competitive with respect todNTPs resulting in increased rates of dNTP incorporation (Fig.3 and Table 1). By increasing reaction rates, SO also promotesincreased misincorporation and misextension by Pol � (Fig. 4,A and B). Furthermore, we show that endogenous Pol � isolatedfrom cells in culture is stimulated by SO similarly as the recom-binant enzyme (Fig. 5, A and B). Notably, we show that 1) SOincreases CPD lesion bypass activity in cells (Fig. 6) and that 2)membrane-bound SO has similar effects as free SO i.e. stimu-lation at low concentrations and inhibition at higher concen-trations (Fig. 7, A and B).
Sphingolipids such as ceramide, SO, and S1P are bioactivesignaling molecules that regulate cellular processes includinggrowth, proliferation, senescence, and apoptosis (31, 36, 37). Ofthese three compounds, only SO uniquely stimulates the activ-ity of Y-family TLS DNA polymerases. Endogenous free SO isgenerated from the hydrolysis of sphingomyelin, which is pres-ent in plasma and nuclear membranes, and subnuclear mem-brane fractions tightly associated with chromatin. It is plausiblethat SO is also anchored to nuclear membrane fractions that arein proximity to sites of DNA transactions involving DNA poly-merases. It has been reported that UV irradiation increases cer-amide levels and, presumably, sphingosine levels by the actionof ceramidase (38). The resulting SO could regulate Pol � activ-ity in a UV and SO dose-dependent manner in vivo.
Sphingosine has been reported to regulate the activity of anumber of protein kinases including PKC, PKB/AKT, MAPK,and v/c-Src kinase (39), all implicated in cell proliferation; ineach case, SO inhibits kinase activity to potentially limit cellproliferation. SO was also shown to inhibit the RNA primaseactivity of the replicative DNA polymerase, Pol (32) andinhibit transcription of CYP17 by binding to the nuclear recep-tor, steroidogenic factor-1, essential for steroid hormone bio-synthesis (40). Our studies on SO are unique in that rather thanthe reported inhibitory effects, we show stimulation of enzymeactivity at intracellular concentrations of SO (32). Interestingly,the expression of Pol � is highest in the adrenal cortex- the siteof steroid biosynthesis (41), and Pol � has also been implicated inbypassing DNA adducts generated during steroid biosynthesis
(42). Thus, SO serves to both regulate steroid hormone synthesisas well as facilitate bypass of resultant steroid adducts by Pol �.
Together with ceramide and S1P, SO is a part of the rheostatthat decides whether cells grow and divide or die. Althoughceramide controls cell growth inhibition and apoptosis induc-tion, S1P contributes to cell proliferation, metastasis, andresistance to apoptosis (37). The effect of SO on the other handdiffers depending on its levels; low levels are reported to pro-mote cell proliferation, whereas high levels are proapoptotic(35). We propose that up-regulation of TLS DNA polymeraseactivity by SO may be necessary to prevent replication forkstalling in response to cellular stress and, therefore, to promotecellular proliferation. It is certainly consistent with our obser-vations where low levels of both free and membrane-bound SOthat stimulate TLS Pol activity promote cellular proliferation,whereas high levels, which are proapoptotic, inhibit activity.
In bacteria, blockage of fork progression induces transcrip-tion of error-prone DNA polymerases. This process, referred toas “the SOS response,” is induced to limit the potential detri-mental effects of fork stalling, i.e. DNA breaks and ensuing rear-rangements and deletions (43). Likewise, there are reports oftranscriptional up-regulation of TLS DNA polymerases inhuman cells (41, 44, 45) as well as evidence for post-transla-tional modification and cellular relocalization upon DNA dam-age (46). Furthermore, there are reports that TLS DNA poly-merase protein levels are elevated in a number of cancers,presumably, to handle the overload of DNA adducts generatedby chemotherapeutic drugs (17, 19, 20). Overexpression oftranslesion DNA polymerases may reduce the effectiveness ofchemotherapy and render cancer cells chemoresistant (21).Modulation of polymerase activity by sphingolipids followingcellular stress may be yet another mechanism by which TLSDNA polymerases is regulated. Our studies provide a novel linkbetween two processes involved in the stress response of cells:error-prone DNA synthesis to avoid replication collapse andsignaling by lipid molecules to control cellular proliferation.
Acknowledgments—We thank Ayu Rahardjo for help in screening thesmall molecule libraries, Drs. Alexey Merz, Margaret Lo, and Sha-rona Gordon for assistance with liposome preparations, and membersof our laboratories for helpful comments and discussions.
REFERENCES1. Guo, C., Kosarek-Stancel, J. N., Tang, T. S., and Friedberg, E. C. (2009)
Y-family DNA polymerases in mammalian cells. Cell Mol. Life Sci. 66,2363–2381
2. Waters, L. S., Minesinger, B. K., Wiltrout, M. E., D’Souza, S., Woodruff,R. V., and Walker, G. C. (2009) Eukaryotic translesion polymerases andtheir roles and regulation in DNA damage tolerance. Microbiol. Mol. Biol.Rev. 73, 134 –154
3. Lange, S. S., Takata, K., and Wood, R. D. (2011) DNA polymerases andcancer. Nat. Rev. Cancer 11, 96 –110
4. Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa,M., Araki, M., Iwai, S., Takio, K., and Hanaoka, F. (1999) The XPV (xero-derma pigmentosum variant) gene encodes human DNA polymerase �.Nature 399, 700 –704
5. Cleaver, J. E., Afzal, V., Feeney, L., McDowell, M., Sadinski, W., Volpe, J. P.,Busch, D. B., Coleman, D. M., Ziffer, D. W., Yu, Y., Nagasawa, H., andLittle, J. B. (1999) Increased ultraviolet sensitivity and chromosomal insta-bility related to P53 function in the xeroderma pigmentosum variant.
Sphingosine Modulates Human TLS DNA Polymerase Activity
AUGUST 1, 2014 • VOLUME 289 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 21671
Cancer Res. 59, 1102–11086. Masutani, C., Kusumoto, R., Iwai, S., and Hanaoka, F. (2000) Mechanisms
of accurate translesion synthesis by human DNA polymerase �. EMBO J.19, 3100 –3109
7. Vaisman, A., Masutani, C., Hanaoka, F., and Chaney, S. G. (2000) Efficienttranslesion replication past oxaliplatin and cisplatin GpG adducts by hu-man DNA polymerase �. Biochemistry 39, 4575– 4580
8. Kino, K., Ito, N., Sugasawa, K., Sugiyama, H., and Hanaoka, F. (2004)Translesion synthesis by human DNA polymerase � across oxidativeproducts of guanine. Nucleic Acids Symp. Ser. 48, 171–172
9. Pavlov, Y. I., Rogozin, I. B., Galkin, A. P., Aksenova, A. Y., Hanaoka, F.,Rada, C., and Kunkel, T. A. (2002) Correlation of somatic hypermutationspecificity and A-T base pair substitution errors by DNA polymerase �during copying of a mouse immunoglobulin � light chain transgene. Proc.Natl. Acad. Sci. U.S.A. 99, 9954 –9959
10. Nair, D. T., Johnson, R. E., Prakash, S., Prakash, L., and Aggarwal, A. K.(2004) Replication by human DNA polymerase-iota occurs by Hoogsteenbase-pairing. Nature 430, 377–380
11. Suzuki, N., Ohashi, E., Kolbanovskiy, A., Geacintov, N. E., Grollman, A. P.,Ohmori, H., and Shibutani, S. (2002) Translesion synthesis by humanDNA polymerase � on a DNA template containing a single stereoisomerof dG-(�)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Biochemistry 41, 6100 – 6106
12. Bi, X., Slater, D. M., Ohmori, H., and Vaziri, C. (2005) DNA polymerase �is specifically required for recovery from the benzo[a]pyrene-dihydrodiolepoxide (BPDE)-induced S-phase checkpoint. J. Biol. Chem. 280,22343–22355
13. Sale, J. E., Lehmann, A. R., and Woodgate, R. (2012) Y-family DNA poly-merases and their role in tolerance of cellular DNA damage. Nat. Rev. Mol.Cell Biol. 13, 141–152
14. Nelson, J. R., Lawrence, C. W., and Hinkle, D. C. (1996) Deoxycytidyltransferase activity of yeast REV1 protein. Nature 382, 729 –731
15. Edmunds, C. E., Simpson, L. J., and Sale, J. E. (2008) PCNA ubiquitinationand REV1 define temporally distinct mechanisms for controlling transle-sion synthesis in the avian cell line DT40. Mol. Cell 30, 519 –529
16. Salehan, M. R., and Morse, H. R. (2013) DNA damage repair and tolerance:a role in chemotherapeutic drug resistance. Br. J. Biomed. Sci. 70, 31– 40
17. O-Wang, J., Kawamura, K., Tada, Y., Ohmori, H., Kimura, H., Sakiyama,S., and Tagawa, M. (2001) DNA polymerase �, implicated in spontaneousand DNA damage-induced mutagenesis, is overexpressed in lung cancer.Cancer Res. 61, 5366 –5369
18. Yang, J., Chen, Z., Liu, Y., Hickey, R. J., and Malkas, L. H. (2004) AlteredDNA polymerase � expression in breast cancer cells leads to a reduction inDNA replication fidelity and a higher rate of mutagenesis. Cancer Res. 64,5597–5607
19. Ceppi, P., Novello, S., Cambieri, A., Longo, M., Monica, V., Lo Iacono, M.,Giaj-Levra, M., Saviozzi, S., Volante, M., Papotti, M., and Scagliotti, G.(2009) Polymerase � mRNA expression predicts survival of non-small celllung cancer patients treated with platinum-based chemotherapy. Clin.Cancer Res. 15, 1039 –1045
20. Teng, K. Y., Qiu, M. Z., Li, Z. H., Luo, H. Y., Zeng, Z. L., Luo, R. Z., Zhang,H. Z., Wang, Z. Q., Li, Y. H., and Xu, R. H. (2010) DNA polymerase �protein expression predicts treatment response and survival of metastaticgastric adenocarcinoma patients treated with oxaliplatin-based chemo-therapy. J. Transl. Med. 8, 126
21. Albertella, M. R., Lau, A., and O’Connor, M. J. (2005) The overexpressionof specialized DNA polymerases in cancer. DNA Repair 4, 583–593
22. Doles, J., Oliver, T. G., Cameron, E. R., Hsu, G., Jacks, T., Walker, G. C.,and Hemann, M. T. (2010) Suppression of Rev3, the catalytic subunit ofPol�, sensitizes drug-resistant lung tumors to chemotherapy. Proc. Natl.Acad. Sci. U.S.A. 107, 20786 –20791
23. Xie, K., Doles, J., Hemann, M. T., and Walker, G. C. (2010) Error-pronetranslesion synthesis mediates acquired chemoresistance. Proc. Natl.Acad. Sci. U.S.A. 107, 20792–20797
24. Schmitt, M. W., Matsumoto, Y., and Loeb, L. A. (2009) High fidelity andlesion bypass capability of human DNA polymerase �. Biochimie 91,1163–1172
25. Kamath-Loeb, A. S., Hizi, A., Kasai, H., and Loeb, L. A. (1997) Incorpora-tion of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases. J. Biol.Chem. 272, 5892–5898
26. Akagi, J., Masutani, C., Kataoka, Y., Kan, T., Ohashi, E., Mori, T., Ohmori,H., and Hanaoka, F. (2009) Interaction with DNA polymerase � is requiredfor nuclear accumulation of REV1 and suppression of spontaneous muta-tions in human cells. DNA Repair 8, 585–599
27. Kamath-Loeb, A. S., Shen, J. C., Schmitt, M. W., and Loeb, L. A. (2012) TheWerner syndrome exonuclease facilitates DNA degradation and high fi-delity DNA polymerization by human DNA polymerase �. J. Biol. Chem.287, 12480 –12490
28. Shen, J. C., Fox, E. J., Ahn, E. H., and Loeb, L. A. (2014) A rapid assay formeasuring nucleotide excision repair by oligonucleotide retrieval. Sci. Rep.4, 4894
29. Seber, G. A. (2003) Linear Regression Analysis, 2nd Ed., Wiley, Boston30. Sasaki, H., Arai, H., Cocco, M. J., and White, S. H. (2009) pH dependence
of sphingosine aggregation. Biophys. J. 96, 2727–273331. Morad, S. A., and Cabot, M. C. (2013) Ceramide-orchestrated signalling in
cancer cells. Nat. Rev. Cancer 13, 51– 6532. Simbulan, C. M., Tamiya-Koizumi, K., Suzuki, M., Shoji, M., Taki, T., and
Yoshida, S. (1994) Sphingosine inhibits the synthesis of RNA primers byprimase in vitro. Biochemistry 33, 9007–9012
33. Zhang, H., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) Sphingosinestimulates cellular proliferation via a protein kinase C-independent path-way. J. Biol. Chem. 265, 76 – 81
34. Zorn, J. A., and Wells, J. A. (2010) Turning enzymes ON with small mol-ecules. Nat. Chem. Biol. 6, 179 –188
35. Ohanian, J., and Ohanian, V. (2001) Sphingolipids in mammalian cell sig-nalling. Cell Mol. Life Sci. 58, 2053–2068
36. Ponnusamy, S., Meyers-Needham, M., Senkal, C. E., Saddoughi, S. A.,Sentelle, D., Selvam, S. P., Salas, A., and Ogretmen, B. (2010) Sphingolipidsand cancer: ceramide and sphingosine-1-phosphate in the regulation ofcell death and drug resistance. Future Oncol. 6, 1603–1624
37. Pyne, N. J., and Pyne, S. (2010) Sphingosine 1-phosphate and cancer. Nat.Rev. Cancer 10, 489 –503
38. Verheij, M., Bose, R., Lin, X. H., Yao, B., Jarvis, W. D., Grant, S., Birrer,M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z.,and Kolesnick, R. N. (1996) Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380, 75–79
39. Mao, C., and Obeid, L. M. (2008) Ceramidases: regulators of cellular re-sponses mediated by ceramide, sphingosine, and sphingosine-1-phos-phate. Biochim. Biophys. Acta 1781, 424 – 434
40. Urs, A. N., Dammer, E., and Sewer, M. B. (2006) Sphingosine regulates thetranscription of CYP17 by binding to steroidogenic factor-1. Endocrinol-ogy 147, 5249 –5258
41. Velasco-Miguel, S., Richardson, J. A., Gerlach, V. L., Lai, W. C., Gao, T.,Russell, L. D., Hladik, C. L., White, C. L., and Friedberg, E. C. (2003)Constitutive and regulated expression of the mouse Dinb (Pol�) geneencoding DNA polymerase �. DNA Repair 2, 91–106
42. Singer, W. D., Osimiri, L. C., and Friedberg, E. C. (2013) Increased dietarycholesterol promotes enhanced mutagenesis in DNA polymerase �-defi-cient mice. DNA Repair 12, 817– 823
43. Goodman, M. F., and Tippin, B. (2000) Sloppier copier DNA polymerasesinvolved in genome repair. Curr. Opin. Genet. Dev. 10, 162–168
44. Lemée, F., Bavoux, C., Pillaire, M. J., Bieth, A., Machado, C. R., Pena, S. D.,Guimbaud, R., Selves, J., Hoffmann, J. S., and Cazaux, C. (2007) Charac-terization of promoter regulatory elements involved in downexpression ofthe DNA polymerase � in colorectal cancer. Oncogene 26, 3387–3394
45. Brauze, D., and Rawluszko, A. A. (2012) The effect of aryl hydrocarbonreceptor ligands on the expression of polymerase (DNA directed) � (Pol�),polymerase RNA II (DNA directed) polypeptide A (PolR2a), CYP1B1 andCYP1A1 genes in rat liver. Environ. Toxicol. Pharmacol. 34, 819 – 825
46. Chen, Y. W., Cleaver, J. E., Hatahet, Z., Honkanen, R. E., Chang, J. Y., Yen,Y., and Chou, K. M. (2008) Human DNA polymerase � activity and trans-location is regulated by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 105,16578 –16583
Sphingosine Modulates Human TLS DNA Polymerase Activity
21672 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 31 • AUGUST 1, 2014
